Project Summary The circadian clock regulates many aspects of life, including sleep and activity and body temperature (BTR) rhythms. We recently identified a novel Drosophila circadian output, temperature preference rhythm (TPR), in which the flies preferred rises in temperature during the day and falls during the night. Our recently published data suggest that fly TPR shares features with mammalian BTR. Drosophila are ectotherms, which typically regulate body temperature behaviorally. Therefore, seeking a preferred temperature is the strategy used to regulate the body temperatures of flies. The circadian clock cells in the fly brain are functional homologs of mammalian SCN (Suprachiasmatic nucleus) neurons. We showed that a small group of circadian neurons, the dorsal neuron 2s (DN2s), specifically regulate TPR, but not locomotor activity, indicating that TPR and locomotor activity are controlled through distinct circadian neurons. Therefore, understanding the TPR will provide new insights into the molecular and neural mechanisms controlling circadian rhythms. The goal of this proposal is to define how neuropeptides regulate TPR and how thermosensory neurons contribute to TPR. We found that PDF (Pigment Dispersing Factor), a critical neuropeptide for locomotor activity, is not involved in TPR, whereas the neuropeptide, DH31 (Diuretic Hormone 31), its receptors DH31R and PDFR and the key clock neurons, DN2s, are necessary for normal TPR. In Aim 1, we will elucidate the mechanisms by which DH31 regulates TPR. In Aim 2, we will examine the mechanisms by which DN2s regulate TPR. Furthermore, our preliminary data suggests that thermosensory neurons are critical for TPR. In Aim 3, we will determine whether thermosensory neurons participate in the neuronal network, controlling TPR.